title: Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5.
abstract: Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.
authors.affiliation: Physiological Laboratory, University of Liverpool, Liverpool, L69 3BX, United Kingdom.
authors.forename: A T
authors.initials: AT
authors.lastname: Jones
authors.forename: I G
authors.initials: IG
authors.lastname: Mills
authors.forename: A J
authors.initials: AJ
authors.lastname: Scheidig
authors.forename: K
authors.initials: K
authors.lastname: Alexandrov
authors.forename: M J
authors.initials: MJ
authors.lastname: Clague
mesh_terms: D019883:Alkyl and Aryl Transferases; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D002460:Cell Line; D002966:Clathrin; D018699:Coated Vesicles; D006224:Cricetinae; D011992:Endosomes / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007668:Kidney; D008561:Membrane Fusion / Q000187:drug effects*; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012265:Ribonucleotides / Q000378:metabolism; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
chemical_list: D019883:Alkyl and Aryl Transferases; D000730:Androstadienes; D002352:Carrier Proteins; D002966:Clathrin; D004791:Enzyme Inhibitors; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012265:Ribonucleotides; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; C065326:xanthosine 5'-triphosphate